(From the Children's Hospital Research Foundation and the Department of Pediatrics, University o/cincinnati College of Medicine, Cincinnati)

Transcription

1 Published Online: 1 December, 1941 Supp Info: Downloaded from jem.rupress.org on July 3, 2018 THE NATURAL HISTORY OF HUMAN POLIOMYELITIS ii. ELIMINATION OF THE VIRUS* BY ALBERT B. SABIN, M.D., AND ROBERT WARD, M.D. (From the Children's Hospital Research Foundation and the Department of Pediatrics, University o/cincinnati College of Medicine, Cincinnati) (Received for publication, July 19, 1941) The purpose of the present investigation was twofold: (1) to determine by which secretions and excretions infective poliomyelitis virus is chiefly eliminated from the human body, and (2) to discover, if possible, the origin of the eliminated virus. This study which was to be carried out on living poliomyelitis patients was planned simultaneously with another investigation the purpose of which was to map the distribution of the virus in the body after death (1). The results of this latter task have been reported (1). At the time the present study was undertaken, these results were not yet available but the following was known about the elimination of virus in human poliomyelitis: (1) that in several hundred tests recorded in the literature the virus had been demonstrated on a number of occasions in nasopharyngeal washings, although the manner in which the tests were performed failed to indicate whether the virus originated from the nose or from the mouth and pharynx, or both; (2) that the virus had been found in the oro-pharyngeal washings on two occasions during the first 2 days of illness in abortive poliomyelitis although similar tests on a few patients with paralytic poliomyelitis were negative (2); (3) that compared with its rare occurrence in nasopharyngeal washings, the virus could be isolated with considerable frequency if not regularly from the stools of patients with abortive, non-paralytic, and paralytic poliomyelitis, and "healthy" contacts as well (3-6). The commonest explanation for the presence of the virus in the stools had been that it had its origin in the swallowed secretions of the upper respiratory tract. The greater incidence of positive isolations from the stools was accounted for by the fact that virus swallowed over a period of days could be concentrated in the colon. When it was recalled that the nasopharyngeal washings represented at best only a casual sample of the secretions, this explanation did not lack plausibility and it appeared possible that if these secretions were collected over a period of days the virus might be demonstrated in them with as great frequency as in the stools. In order to test this hypothesis, * Aided by a grant from the National Foundation for Infantile Paralysis, Inc. 519

2 520 NATLrRAL HISTORY OF HUMAN POLIOMYELITIS. II and without any preconceived plan of separating the nasal from the pharyngeal and oral secretions, the nasal secretions were collected by alternately plugging one or the other side of the nose with absorbent cotton for a period of 3 days; at the end of this period a stool or enema returns specimen was obtained to serve as ~n index to the presumable accumulation of virus in the lower intestine during the interval under investigation. Whenever possible, and that, unfortunately, was usually only among the older patients, the oral secretions and saliva were also collected during the same period. The urine of a number of patients, particularly when clinical signs of bladder involvement were present, was studied to test the hypothesis that poliomyelitis virus may spread centrifugally from the spinal cord along certain nervous pathways to the intestines and urinary bladder and ultimately be eliminated from these sites. Collection of Specimens and Methods of Preparation The material for this study was collected in the summer of 1940 from patients in Jackson, Michigan, Indianapolis, and Cincinnati. 1 Nasal Secretions.--Absorbent cotton was plugged alternately into one or the other side of the nose every 6 to 18 hours for a period of 3 days. As each plugwas removed (usually soaked with secretions) it was stored in a refrigerator. At the end of the 3 days, the 6 plugs usually obtained from each patient were pooled and transported to the laboratory in the frozen state (solid CO2 was used). The secretions were expressed from the plugs in several steps: (1) approximately 4 to 7 cc. of undiluted secretion was obtained by compressing the plugs in a syringe containing a piece of wire gauze over its floor; this specimen was centrifuged at about 2,000 R.P.M. for 10 minutes, the sediment being saved for nasal instillation while the supematant liquid after etherization was used in part for intracerebral inoculation and the remainder combined with the second extract for intraperitoneal inoculation; (2) the second extract was obtained by macerating the plugs in 5 to 10 cc. of physiological salt solution and again expressing the fluid with the aid of a syringe; after centrifugation, as above, the sediment was saved for nasal instillation, and the supematant liquid, after etherization, for intraperitoncal inoculation; (3) the fluid obtained by extracting the plugs with another 10 co. of physiological salt solution was added without centrifugation or etherization to the two previous sediments and used for nasal instillation. The etherized specimens were prepared by adding 15 per cent of anesthetic ether, shaking for I0 minutes, storing in the refrigerator overnight, and then centrifuging twice at about 2,000 R.P.~. The various extracts were inoculated into the same monkey intracerebrally (under local anesthesia), intraperitoneauy, and intranasally. Stools or Enema Returns.--When a stool could not be obtained at the end of the period of collection of nasal and oral secretions, the returns from a physiological salt solution enema were used. The amount of solid matter in the enema returns may not be important, since on several occasions we were able to find the virus in enema fluid that was only slightly tinged with brown. When solid fecal material was available it was thoroughly ground in sufficient physiological salt solution, distilled water, or, 1 We wish to thank Dr. Frank Van Schoick of Jackson, Michigan, and Dr. Lyman T. Meiks and Dr. Benjamin Siebenthal of Indianapolis for their cooperation.

3 ALBERT B. SABIN AND ROBERT WARD 521 whenever present, in enema fluid to make a 10 or 20 per cent suspension. The preparation of these suspensions for inoculation was the same as that described in a previous publication for the colon contents (1). The methods we used in demonstrating the virus were influenced largely by the results reported by Trask, Paul, and Vignec (3) and by those of Howe and Bodian (6). Paul, Trask, and Gard (7) observed that they lost very few monkeys when they limited their inoculations of etherized material to the intraperitoneal route and when the amount in a single inoculum did not exceed 20 cc. Using this method for the most part, the total number of positive virus isolations (15 per cent of 53 patients) reported by Trask, Paul, and Vignec (3) was, however, rather low. Howe and Bodian (6), on the other hand, reported the demonstration of virus in 10 of 14 patients (i.e. about 70 per cent) by simple nasal instillation of the untreated stools. We compared the two methods on the first five specimens we obtained (Table I) and found that in this small test, at least, the intraperitoneal TABLE I Comparison of Intraperitoneal and Intranasal Mahods for Dejection of Poliomyditis Virus in Same Specimen of Stool Patient Wal. Me G. V.d.B. Gil. Kee. Age yes. 3½ Centrifuged, etherized portion intraperltoneally Total dose cc. Result 38 P 39 P O P = paralytic poliomyelitis in inoculated monkey. N P = non-paralytic poliomyelitis in inoculated monkey. Untreated portion intraaasally Total dose cc. Result 20 N P method was superior to the intranasal although it was evident that nasal instillation of untreated stool can give rise to poliomyelitis infection. We, therefore, combined the two methods with the modification that while a single intraperitonealinoculum did not exceed 20 cc., each monkey was to receive at least an additional 20 cc. intraperitoneally 24 to 48 hours after the first injection whenever enough material was available. 2 A portion of the untreated stool (when the specimen was large enough) Since submitting this paper for publication we have obtained convincing evidence that in cynomolgus monkeys the upper as well as lower parts of the alimentary canal can serve as portals of entry for the virus. Because after nasal instillation of untreated stools or other contaminated specimens, most of the material becomes distributed throughout the alimentary tract we now prefer to use cynomolgus rather than rhesus monkeys, since in the latter only the olfactory mucosa seems to function as a portal of entry for the virus. Using the combined nasal instillation of the untreated portion and intraperitoneal injection of the etherized part of the specimen in cynomolgus monkeys, three successive enema returns obtained on the 2nd, 4th, and 6th days of paralysis from a 35 year old patient yielded the virus.

4 522 NATURAL HISTORY OF HUMAN POLIOMYELITIS. II or the resuspended centrifuged sediment (after removal of the supernatant liquid for etherization) was given intranasaiiy--1 ec. in each nostril daily for 10 d~ys or less if there was not enough material; most monkeys received between 10 and 20 cc. in this manner. In a number of monkeys, following the procedure of Howe and Bodian (6), the nasal passages were gently rubbed with a pipe cleaner immediately after the instillation of stool, but one cannot be certain that this increased the incidence of infection by way of the nose. Table II gives a summary of the methods and materials used, and the results obtained. The olfactory bulbs and often also the anterior perforated substance were examined in all monkeys and lesions were regularly absent TABLE II Isolation of Poliomyelitis Virus by Various Methods from Different Types of Lower Intestinal Contents Method of testing for virus One i.p... Two i.p... One i.p. -b i.nas. (no p.c.) Two i.p. -b i.nas. (no p.c.) One i.p. -b i.nas. (p.c.).. Two i.p. -b i.nas. (p.c.). Stool 1/4. 2/5 [0,?lt 0/1 1/3 [2] Type of lower intestinal contents 0/2 1/1 Enema returns 1/3 [o1 3/4 [o, o, +1 Descending colon contents from fatal cases 2/2 I/I [q-i 4/4 [+, +, +, 0] Total... 4/13 (31 per cent) 5/10 (50 per cent) 7/7 (100 per cent) i.p. = intraperitoneal; Lnas. --- intranasal; p.c. = pipe cleaner used after nasal instillation of specimen. "1/4 -- virus isolated from one of four specimens. t [0,?] = data in brackets refer to result of examination of olfactory bulbs of monkeys succumbing with paralysis; in this instance, it means that no lesions were found in the bulbs of one monkey and that in the other they were either not examined or that the sections were unsatisfactory. [0, O, q-] -- olfactory bulb lesions absent in 2 monkeys and present in one. when poliomyelitis developed following only intraperitoneal inoculation; the presence of lesions in these sites in some of the monkeys receiving the combined intranasal and intraperitoncal inoculations indicates that in some of the animals the virus invaded the nervous system from the nose by the olfactory pathway. However, the fact that in other paralyzed monkeys which received the combined inoculations, no lesions were present in the olfactory bulbs suggests that in these monkeys infection resulted from the material injected in the peritoneal cavity, and supports the conclusion drawn from the comparative test (Table I) that it is inadvisable to rely on the intranasal method as the sole means of detecting virus. Saliva and Oral Seeretions.--Patients, 9 years of age and older, supplied specimens by expectorating into bottles or Petri dishes over a period of 3 days. During this time the secretions were transferred at various intervals to a sterile rubber-stoppered container which was kept in the refrigerator. The total amount collected from

5 ALBERT B. SABIN AND ROBERT WARD 523 different patients varied between 6 cc. and 75 cc. and in most instances was 20 cc. or more. A portion of the saliva and oral secretions, including the centrifuged sediment, was untreated and instilled intranasally into a monkey while the remainder was etherized and prepared in the same manner as the nasal secretions, and inoculated intracerebrally and intraperitoneally into the same animal. Urine.--Urine was obtained by catheterization from 12 patients, 6 of whom had paralysis of the bladder. In only 7 instances, however, was it possible to inject the untreated urine into monkeys; in 2 of these, 200 cc. was injected intraperitoneauy in 4 divided doses (50 cc. in the morning, and 50 cc. in the late afternoon) over a period of 2 days, in 2 others 120 cc. to 150 cc. was given, and in the remaining 3 cases, 80 to 100 cc. was administered in the same manner in 4 divided doses. Because they contained too many bacteria, each of the other specimens was divided into two portions, one of which was filtered through a Berkfeld "V" filter and the other etherized. 100 cc. of each of 3 such specimens were injected intraperitoneally into monkeys. In only 2 instances were smaller amounts (35 cc. and 50 cc. respectively) inoculated. Animals, Observations, and Criteria for Diagnosis of Experimental Poliomyelitis.-- Rhesus monkeys were used and the criteria for positive isolation of poliomyelitis virus were described in the previous communication (1). RESULTS The nasal secretions obtained from 22 paralyzed patients during the first 2 weeks of the disease did not yield the virus in a single instance, while in 9 of the same 22 patients the stools or enema returns, collected as previously described, contained readily demonstrable poliomyelitis virus. The nasal secretions of three 5 to 6 year old children with non-paralytic poliomyelitis were studied in the same manner with negative results, but tests for virus in the stools of two of these patients were also negative. No virus was found in the saliva and oral secretions of 20 patients, but in only 10 of them were tests made on the lower intestinal contents, and three of these yielded the virus. Tests for virus in large amounts of urine from 12 paralyzed patients were also all negative, but no simultaneous studies were made on the stools of these patients. Table III contains the data on the tests for virus in the secretions of the 10 paralyzed patients whose lower intestinal contents contained the virus. The results do not support the hypothesis that the virus in the stools has its origin in swallowed nasal secretions and saliva, but are in agreement with our findings on the distribution of virus in fatal cases of human poliomyelitis in which virus was demonstrated in the walls of the pharynx and intestines, but not in the nasal mucosa or salivary glands (1). It is not improbable, therefore, that in the valid reports of isolation of virus from nasopharyngeal washings, the virus may have originated from the pharyngeal wall rather than from the nose. The incidence of 9 positive isolations of virus from the stools of 23 paralytic patients (Table IV) is higher than that (4 out of 38) reported for paralytic cases by Trask, Paul, and Vignec (3) but less than that (10 out of 14) obtained by Howe and Bodian (6). The former investigators (3) were impressed with

6 524 NATURAL HISTORY OF M-UA~AN POLIOMYELITIS. II ~ o ~W ~z = O0 ~'~ e~ T T T O.o O =o O O ~o t~ 4- o e~,6

7 ALBERT B. SA.BIN AND ROBERT WARD 525, ~ ~ ~ ~ ~ + ~ +? = + = t ,.+, 0 ~ - rj ci o~ "+.++ P~ m+ ~u- o ~++ "m mll~e ~ + ] + os.+ +~,, )~ ++' 1:::1 +'~ f ~0 ~ m m i~ <:+<- il H g 11 11

8 526 NATURAL HISTORY OF B'U]~AN POLIOMYELITIS. II the fact that with one exception their positive stools were from children 6 years of age or younger. Examination of our own data revealed that while 7 of 11 (64 per cent) fecal specimens were positive in the age group of 5 years and under, only 2 of 12 (17 per cent) were positive in the older patients (one was 13 and the other 22 years old). However, when our data were analyzed statisti- TABLE IV Excretion of Virus during the First 2 Weeks of Human Paralytic Pollomyditis Material tested No.?f [ speclmeils (pa- tients) I Stool or enema returns. 23 Nasal s e 3 days.t 22 secretions (saliva) [ 3 days Urine ( cc.) No. positive 9 (39 per cent) 0 * X 2 with Yates adjustment for small numbers P is between 0.10 and R61e of age of patient--no, positive 5 yrs. and under 7/11" (64per cent) o/11 Over 5 yrs. 2/12" (17 per cent) 0/11 TABLE V Rdation between Age of Patient and Incidence of Positive Isolations of Poliomyditis Virus from Stools 0/20 Source of data Kramer, Hoskwith, and Grossman (5)... Kramer, Gilliam, and Molner (4) (contacts--no clinical "polio")... Howe and Bodian (6)... Trask, Paul, and Vignec (3)... Sabin and Ward... Virus isolated from stools of patients under 8 years 2/4 (50 per cent) 5/11 (45 per cent) 8/11 (73 per cent) 7/21 (33 per cent) 7/11 (64per cent)... 8 years and over 1/5 (20 per cent) 1/8 (12 per cent) 2/3 (67 per cent) 1/32 (3 per cent) 2/12 (17 per cent) Total... 29/58* (50 per cent) 7/60* (12 per cent) * x 2 = 20.44; P < cally* it was found that Chi square with the Yates adjustment for small numbers was only 3.29 with a value for P between 0.10 and 0.05, and, therefore, statistically not significant. But when we combined them with the data which, in our opinion, could be justifiably selected from the literature, a marked difference in the incidence of positive stools was evident when the patients were divided into 2 groups--one under 8 years and the other 8 years and older (Table V). In the first group 50 per cent of 58 individuals yielded positive We are indebted to Mrs. Estelle W. Brown for the statistical analysis of our data.

9 ALBERT B. SABIN AND ROBERT WARD 527 stools while in the second group only 12 per cent of 60. Statistical analysis shows this to be a most significant difference with a Chi square value of and P less than One possible explanation of this difference may be that multiplication of the virus is generally more extensive in the younger children. It is of interest in this respect that in contrast to the relative rarity of positive virus isolations from the stools of individuals over 8 years of age, the virus was present in the contents of the descending colon in each of 5 fatal cases in this age group (Table VI). This is a statistically significant difference, and one is naturally led to wonder whether proliferation of the virus is generally more extensive in patients with a fatal termination regardless of age. It is to be recalled, however, that explanations based on unproved assumptions are often wrong, and further investigation of the meaning of these significant differences is indicated. TABLE VI Occurrence of Poliomyelitis Virus in Stools during Life and in Colon Contents Postmortem among Paralytic Patients over 8 Years of Age Material No. of patients Stool or enema returns of living paralytic patients Contents of descending colon postmortem... 5 x 2 with Yates adjustment for small numbers = P is between 0.01 and DISCUSSION No. positive 2 (16.7 per cent) 5 (100 per cent) In a search for the avenues of elimination of infective virus during the first 2 weeks of paralytic human poliomyelitis, it was found that while the virus could be demonstrated with considerable regularity in the stools (notably in children under 8 years of age), it was not obtained in a single instance from the nasal (not nasopharyngeal) secretions or oral secretions and saliva collected over a period of 3 days, or from large amounts of urine. In the light of these findings, the hypothesis that virus is present in the stools because it has been swallowed with nasal or other upper respiratory secretions, becomes untenable, and in view of the demonstration that it is present in the walls of the alimentary tract (1) its origin from these sites is much more probable. It may be suggested, however, that the negative results obtained with the nasal secretions may be due to the presence of an inactivating substance and need not necessarily indicate the absence of virus. If there is such a substance in patients with poliomyelitis it might also be suggested that it should either have been effective in preventing local multiplication in the nasal mucosa, or if it arises in response to infection (and there is no evidence for that assumption) (8) that its presence would thus militate against the spread of infective virus by means of the nasal secretions. However, our studies on the pattern of virus

10 528 NATURAL I:ZISTOR OF II%IMAN POLIOMYELITIS. 211 distribution in the human body (1) suggest that the virus neither multiplied in nor invaded the nervous system by way of the nose, because, while it was readily demonstrated in the pharyngeal and intestinal tissues, it was present neither in the nasal mucosa nor in the olfactory bulbs of the cases that were studied. For practical purposes, therefore, it would appear that in human paralytic poliomyelitis the secretions, as they are expelled from the nose or expectorated from the mouth, are probably, as a rule, free from infective virus. However, since the virus has been demonstrated in the pharyngeal tissue postmortem as well as in a certain number of washings obtained by techniques which involved lavage of the pharyngeal wall, it is necessary to determine whether virus that may be present in the pharynx would under natural conditions be swept into the esophagus or might also find its way into the oral secretions and be expelled with them. Since we have recently observed (unpublished data) that in cynomolgus monkeys which had developed poliomyelitis after oral feeding of the virus, one can find virus in appreciable amounts in the buccal mucosa and tongue as well as ~n the pharyngeal wall and washings of the oro-pharyngeal cavity, it would be well to await similar tests on human beings postmortem as well as further studies on oral washings particularly during the first days of the disease in young children, before attempting to form any concept concerning the r61e of the mouth as a site of exit of virus. Although our tests with large amounts of urine were all negative and while the pattern of virus distribution in the human body indicates that there is not sufficient centrifugal progression of virus to permit its invasion of the urinary bladder, it, nevertheless, seems advisable to await actual tests with bladder tissue and further studies on children under 8 years of age before reaching a final decision about the non-infectiousness of urine. SUM~IARY AND CONCLUSIONS Studies on the elimination of virus in human paralytic poliomyelitis during the first 2 weeks of the disease, revealed the following:-- 1. The nasal (not nasopharyngeal) secretions collected from 22 patients on cotton plugs over a period of 3 days and the saliva and oral secretions expectorated during a similar period by 20 patients failed to yield virus. 2. In 10 of the patients whose secretions (nasal, oral, or both) were investigated, virus was isolated from single specimens of the lower intestinal contents. 3. No virus was found in large amounts of urine (up to 200 cc.) obtained from 12 patients, 6 of whom had paralysis of the bladder. 4. In the present tests virus was found 4 times more often in the stools of patients under 8 years of age (64 per cent of 11 cases) than in older individuals (17 per cent of 12 cases). This difference was found to obtain when our data

11 ALBERT B. SABIN AND ROBERT WARD 529 were combined with those which could justifiably be selected from the literature, the total figures indicating that virus has been isolated from 50 per cent of 58 children under 8 years of age and from 12 per cent of 60 older individuals. 5. No support was found for the hypothesis that poliomyelitis virus in the stools originates from swallowed nasal secretions and saliva or oral secretions. BIBLIOGRAPHY 1. Sabin, A. B., and Ward, R., J. Exp. Med., 1941, 73, Paul, J. R., and Trask, J. D., J. Exp. Med., 1932, 56, Trask, J. D., Paul, J. R., and Vignec, A. J., J. Exp. Med., 1940, 71, Kramer, S. D., GiUiain, A. G., and Molner, J. G., Pub. Health Rep., U. S. P. H. S., 1939, 54, Kramer, S. D., Hoskwith, B., and Grossman, L. H., J. Exp. Med., 1939, 69, Howe, H. A., and Bodian, D., J. Infect. Dis., 1940, 66, Paul, J. R., Trask, J. D., and Gard, S., J. Exp. Med., 1940, 71, Howitt, B. F., J. Infect. Dis., 1937, 60, 113.